The presence of clonally expanded T cells and B cells with their secreted antibody products in the CSF and inflammatory plaque tissue of patients with multiple sclerosis (MS) remains a major hallmark of the disease. Yet in MS, the central question of whether there are specific antigen reactivities of clonally expanded T or B cells in brain plaques has not been addressed. Moreover, our recent examination of autoantibodies during the last application period has unexpectedly led us to observe that antigen specificity of CSF or serum IgG does not reflect the specificity of IgG isolated from CNS plaques. Thus, direct examination of the inflamed MS plaques particularly in relation to the newly recognized patterns of MS pathology is critical. We will interrogate individual single cells in plaque tissue examining CD4+ and CD8+ cells for transcription factors and newly described cell surface molecules such as Tim-3 that define immune function in relationship to the lesion's pathologic pattern. We hypothesize that the different pathologic subtypes of MS will be linked with different immunopathological processes mediated by different T cell functions. Specifically, we postulate that the pathologic pattern I which shows close similarities to T-cell-mediated pathophysiology will have a more Th1 cytokine balance, while pattern II which appears to be more like T-cell plus antibody-mediated autoimmune encephalomyelitis will exhibit a different cytokine pattern. Moreover, the application of laser-microdissection and single cell PCR on MS plaque sections will allow us to produce large amounts of recombinant immunoglobulin from oligoclonal B cells. These rare immunoglobulins will be used for antigen identification by a variety of sensitive molecular techniques including mass spectrometry and use of the Harvard Full Length Expression Library derived from the entire human genome. In the last aim, a panel of TCRs derived by single cell cloning of CSF T cells and by single cell PCR from clonally expanded TCR sequences from brain tissue from MS plaques and other inflammatory CNS diseases will be examined for reactivity to combinatorial peptide will focus on the inflammatory demyelinating tissue and will examine the immunologic basis of the newly described pathologic subpatterns of MS in addition to exploring, without bias, the antigens driving the T and B cell clonal expansion.